Chemical insecticides are an integral component of modem agriculture, and have afforded an effective means for reducing crop damage by controlling insect pests. However, chemical agents are under continuous scrutiny due to the potential for environmental contamination, selection of resistant populations of agronomic pests, and toxicity to non-target organisms such as beneficial insects, aquatic organisms, animals and man. As a result, alternative strategies for insect control are being sought that are effective and yet benign to non-target populations and the environment. One of these strategies comprises the use of microorganisms that are naturally occurring pathogens of target insect populations. However, many candidate entomopathogens that would be promising insect control agents lack the properties of classical chemical insecticides that farmers and others in agribusiness have grown accustomed to. For instance, insect-specific viruses from the family Baculoviridae possess several favorable attributes, including host-specificity and inert environmental properties, but lack the ability to rapidly control a target population before significant crop damage takes place. Fortunately, modern molecular biology provides the tools necessary to favorably modify many of these properties in order to satisfy the needs of modern agriculture.
Baculoviruses are viruses pathogenic to invertebrates, and are characterized by possession of a double-stranded, circular DNA genome ranging in size from 80 to 200 kilobases. Baculoviruses are divided into three subfamilies, including non-occluded baculoviruses (NOVs), granulosis viruses (GVs) and nuclear polyhedrosis viruses (NPVs). Examples of NOVs are Orcytes rhinoceros NOV and Helicoverpa zea NOV. Examples of GVs include Plutella xylostella GV, Cydia pomonella GV, Pieris brassicae GV, and Trichoplusia ni GV. Examples of NPVs include Autographa californica NPV, Spodoptera exigua NPV, Heliothis armigera NPV, Helicoverpa zea NPV, Spodoptera frugiperda NPV, Trichoplusia NPV, Mamestra brassicae NPV, Lymantria dispar NPV, Spodoptera litturalis NPV, Syngrapha facifera NPV, Choristoneura fumiferana NPV, Anticarsia gemmatalis NPV, and Heliothis virescens NPV.
Although certain GVs and NOVs have been carefully studied, NPVs are the most thoroughly characterized of the baculovirus subfamilies. The infection cycle of NPVs involves two types of virions. Following infection of insect cells, budded virions (BVs or extra cellular virus, ECV) are produced upon movement of nucleocapsids to the plasma membrane. These virions shed their nuclear-derived coat in the cytoplasm and bud through the cytoplasmic membrane into the hemocoel of the insect host. This process leads to systemic infection of the host insect. Later in the infection process, virions become occluded (occluded virions) within a protein matrix consisting substantially of the polyhedrin protein, thus forming polyhedrin inclusion bodies (PIBs or occlusion bodies, OBs). These inclusion bodies are the orally infectious form of the virus, and provide for horizontal transmission of the virus between insect hosts (1,2). Uninfected larvae feed on virus-contaminated substrates and ingest PIBs. The proteinaceous matrix is solubilized by the action of the basic pH of the insect midgut found in many lepidopterous larvae. The liberated virion nucleocapsids, containing the viral DNA genome, attach to and infect the epithelial cells of the larval midgut. Typically, the infected insect will continue to develop and consume plant material while the virus exponentially propagates within the host. Eventually, often after several weeks or longer have passed, the infected larvae will become filly involved and expire.
An attractive attribute of baculoviruses is their narrow host specificity. These viruses infect only arthropods, and possess relatively narrow host ranges even within a particular insect order. Host specificity has been examined by electron microscopy, DNA hybridization and recombinant DNA technology (3-5). These studies indicate that the narrow host range is due, at least in part, to the inability of baculoviruses to transfer viral DNA into the manalia cell nucleus.
Due in part to the availability of efficient cell culture systems and facile cloning vectors, NPVs have been utilized as eukaryotic expression vectors for synthesis of desirable heterologous proteins (6,7). One virus in particular, Autographa californica NPV (AcNPV), is the accepted model virus utilized for introduction and expression of heterologous genes in baculovirus expression systems. Although this virus is routinely used as an important in vitro means of providing for high yields of recombinant proteins in a eukaryotic expression system, thus affording appropriate post-translational modification of expressed proteins, AcNPV is capable of infecting many families of Lepidopteran insects that are important economic pests.
In spite of the potential practical advantages of baculovirus-based pest control agents, a variety of disadvantages have curtailed their use in modern agriculture. The most significant barrier to more widespread use of these viruses in row-crop agriculture is the significant time delay between their application and effective control of crop damage caused by the host insects. Unlike the rapid effects observed upon application of classical chemical insecticides, effective wild-type baculovirus-mediated insect control occurs only after in vivo populations of virus have reached levels high enough to compromise host activity. However, through the use of recombinant DNA technology, NPVs have been genetically engineered to increase their rate of insect killing by either the introduction of genes directing the expression of insecticidal proteins, or deletion of genes from the viral genome (8-10). The most effective recombinant NPVs have been engineered to express insect-selective neurotoxins (11-18). These recombinant viruses kill their hosts in 20-30% less time than wild-type NPVs.
There has now been constructed recombinant NPVs that have significantly greater potency than previously constructed recombinant NPVs. These recombinant NPVs have been engineered to express a heterologous gene encoding the insect-selective toxin LqhIT2 of the scorpion Leiurus quinquestriatus hebraeus (19,20). Based on present studies, the recombinant NPVs carrying this synthetic gene provide for a significant increase in the insecticidal properties of the virus.